Type 2 isopentenyl diphosphate isomerase from a thermoacidophilic archaeon Sulfolobus shibatae

Abstract

Although isopentenyl diphosphate-dimethylallyl diphosphate isomerase is thought to be essential for archaea because they use the mevalonate pathway, its corresponding activity has not been detected in any archaea. A novel type of the enzyme, which has no sequence similarity to the known, well-studied type of enzymes, was recently reported in some bacterial strains. In this study, we describe the cloning of a gene of a homologue of the novel bacterial isomerase from a thermoacidophilic archaeon Sulfolobus shibatae. The gene was heterologously expressed in Escherichia coli, and the recombinant enzyme was purified and characterized. The thermostable archaeal enzyme is tetrameric, and requires NAD(P)H and Mg 2+ for activity, similar to its bacterial homologues. Using its apoenzyme, we were able to confirm that the archaeal enzyme is strictly dependent on FMN. Moreover, we provide evidence to show that the enzyme also has NADH dehydrogenase activity although it catalyzes the isomerase reaction without consuming any detectable amount of NADH.Keywords: isopentenyl diphosphate-dimethylallyl diphosphate isomerase; isoprenoid; archaea; flavoprotein; NADH dehydrogenase.Isoprenoid compounds are the most diverse family of metabolites found in nature. They are necessary for all living organisms because they are functional parts of important compounds, including vitamins, hormones, respiratory quinones, and archaeal membranes [1]. The majority of isoprenoid compounds are synthesized from linear prenyl diphosphates, which are formed via the consecutive condensation of isopentenyl diphosphate (IPP), the active isoprene C 5 -unit, to its highly electrophilic isomer dimethylallyl diphosphate (DMAPP).Isopentenyl diphosphate-dimethylallyl diphosphate isomerase (IPP isomerase; EC 5.3.3.2) catalyzes the interconversion of IPP and DMAPP and is a key enzyme in the biosynthesis of isoprenoids [2]. Based on studies using eukaryotes, IPP has been shown to be synthesized from acetyl-CoA via the well-known mevalonate pathway and is further converted to DMAPP by IPP isomerase [3]. On the other hand, many bacteria, green algae, and chloroplasts of higher plants have recently been shown to use a different isoprenoid biosynthetic pathway, which is referred to as the nonmevalonate pathway [4,5]. It has been reported that IPP and DMAPP are synthesized separately in Escherichia coli and that the IPP isomerase gene was not essential for this organism [6]. Synechocystis sp. strain PCC6803, which also utilizes the nonmevalonate pathway for the biosynthesis of isoprenoids, was shown to be deficient in IPP isomerase activity [7]. In short, IPP isomerase is necessary for the biosynthesis of isoprenoid compounds via the mevalonate pathway, and unnecessary for that of the nonmevalonate pathway. However, despite the sole utilization of the mevalonate pathway for isoprenoid biosynthesis, many archaea and some bacteria lack homologues of IPP isomerase genes in their genome sequences [8].Kaneda et al. recently cloned the gene fni from Streptomyces sp. strain C...

New Role of Flavin as a General Acid-Base Catalyst with No Redox Function in Type 2 Isopentenyl-diphosphate Isomerase

“…In all crystal types, the asymmetric unit contains four monomers that are related by a non-crystallographic 4-fold rotation, which is regarded as the tetrameric state of S. shibatae IDI in solution (18). As shown in Fig.…”

“…The transformant was cultivated in L medium containing 50 mg/liter of ampicillin until cells reached the early stationary phase. The recombinant enzyme was purified with heat treatment at 55°C for 30 min and a HisTrap column (GE Healthcare) as described previously (18). For crystallization, the enzyme was loaded on a HiLoad 16/60 Superdex 200 column (GE Healthcare) and eluted with 10 mM Tris-HCl (pH 7.7), containing 1 mM EDTA, 10 mM ␤-mercaptoethanol, and 0.15 M NaCl.…”

“…Enzyme Purification-The plasmid for the expression of (His) 6 -tagged S. shibatae IDI, pET-idi (18), was introduced into the Escherichia coli BL21(DE3) strain. The transformant was cultivated in L medium containing 50 mg/liter of ampicillin until cells reached the early stationary phase.…”

Evidence for the Involvement of Acid/Base Chemistry in the Reaction Catalyzed by the Type II Isopentenyl Diphosphate/Dimethylallyl Diphosphate Isomerase from Staphylococcus aureus

“…CL190, IDI-2 enzymes have been found in many eubacteria and archaebacteria as part of either the MVA or the MEP biosynthetic pathways (13-15). Subsequent biochemical and structural characterization of [15][16][17][18][19][20][21][22] has revealed that the IDI-2-FMN ox complex is reduced with stoichiometric amounts of NAD(P)H via stereospecific transfer of the pro-S hydride and that the resultant IDI-2-FMN red is capable of performing multiple turnovers (21). Recent redox titrations of IDI-2 from Staphylococcus aureus and Thermus thermophilus in the presence and absence of IPP (1) have shown that the apoenzyme thermodynamically stabilizes the neutral flavin semiquinone in the presence of IPP (21,22).…”

“…Furthermore, they suggested that the enzyme-bound FMN intermediate that forms during the normal reaction is consistent with a neutral reduced dihydroflavin (4, Scheme 2). Although the steady-state kinetic parameters have been reported for a few IDI-2 enzymes (13,15,18,21,22,25), the nature of the rate limiting step(s) in the catalytic mechanism is unknown. In this paper, we report the investigation of the FMN intermediate formed during the catalysis of S. aureus IDI-2 using UV-vis and EPR spectroscopies.…”